Project Summary Our understanding of the molecular mechanisms of fusion between reproductive cells (gametes) during sexual reproduction is surprisingly limited. In studies with the unicellular green alga, Chlamydomonas and the malaria pathogen Plasmodium, the Snell laboratory has shown that the broadly conserved, gamete-specific membrane protein HAP2 (also called GCS1) is essential for bilayer merger during gamete fusion. HAP2 acts after tight adhesion of gamete plasma membranes, and is present on only one of the two gametes (e.g., mating type minus gametes in Chlamydomonas) and therefore functions unilaterally, as do viral fusion proteins during their entry into host cells. Remarkably, recent collaborative structure/function studies demonstrated that Chlamydomonas HAP2 is structurally homologous to the class II fusion proteins of many enveloped viruses, including dengue and Zika. Though recombinant forms of the ectodomain of Chlamydomonas HAP2 form trimers in vitro and the hydrophobic fusion loop is essential for function in vivo, we still know very little about the molecular mechanism of this eukaryotic class II fusion protein during gamete fusion in any HAP2 organism. Endogenous trimers that presumably form during gamete fusion have yet to be detected, and the mechanisms are unknown that restrict triggering of trimer formation until the HAP2-bearing gamete undergoes specific membrane interaction with the membrane of its partner gamete. In preliminary experiments, I have discovered a stable oligomer, which appears only after gamete fusion, and is likely to be the endogenous HAP2 trimer. I have also determined that, unlike with the viral class II proteins, low pH fails to trigger oligomer formation of HAP2. Rather, the HAP2-containing minus mating structure must bind to the adhesion protein FUS1 on the plus mating structure, ensuring that triggering takes place only at the right time and place. This research aims to define the molecular mechanisms that underlie the function of a broadly conserved eukaryotic membrane fusogen that is essential for fusion of gametes in organisms across kingdoms. The insights gained from this study will improve our understanding of fundamental, conserved mechanisms of gamete fusion, and have the potential to yield improved strategies for interfering with sexual reproduction and transmission of organism that harm humans, including the devastating malaria organism Plasmodium. Here, I will test the model that a new HAP2 oligomer detected in gametes after fusion is a trimer. I will investigate structural features of HAP2 required for oligomerization. And, I will investigate the mechanisms that underlie FUS1-dependent triggering of HAP2 structural rearrangements.